Glutathione (GSH) is a low molecular weight intracellular thiol that is important in free radical scavenging, detoxification of xenobiotics, protein synthesis, DNA synthesis and repair, and cell proliferation. One consequence of exposure to a wide variety of xenobiotics, is the induction of glutathione biosynthetic enzymes, especially gamma-glutamylcysteine synthetase (GCS), the rate limiting enzyme in GSH biosynthesis. GCS is composed of heavy and light subunits, the former having all the catalytic activity of the enzyme, and the latter possessing features important in regulating certain aspects of GCS activity. Ongoing research has shown variability among humans with respect to induction of GCS after exposure to glutathione depleting drugs. Whether these differences in GCS activity and expression among individuals are attributable to primary differences in the amino acid sequence, or to transcriptional, translational or postranslational modifications are yet to be determined. The primary goal of this proposal will be to investigate the genetic and biochemical bases of GCS regulation in mice and humans. We will test two hypotheses relevant to these goals. The first hypothesis is that exposure of cells and/or animals to glutathione depleting xenobiotics will result in an increase in the mRNA levels for one or both of the GCS subunits, which will be accompanied by an increase in GCS activity and GSH levels. The second hypothesis is that DNA sequence regulatory elements in the 5' region of mouse GCS genes confer tissue-specific regulation of the basal and inducible levels of the heavy and light subunit GCS mRNAs. The third hypothesis is that individual variation among humans in GCS activity and inducibility may be partially attributable to genetic polymorphisms in the 5' regulatory regions of either the heavy or light subunit genes of GCS. The research proposed in this project uses molecular, genetic and cellular approaches to address the questions raised by these hypotheses. Information gathered during the pursuit of these questions will lead to a better understanding of the molecular and cellular bases of glutathione regulation, and such information will be useful not only as a biomarker of exposure to toxicants, but potentially as a biomarker of susceptibility to such exposures.